DETECTION OF ENTEROVIRUSES AND RHINOVIRUSES IN CLINICAL SPECIMENS BY PCR AND LIQUID-PHASE HYBRIDIZATION

A sensitive method based on PCR followed by liquid-phase hybridization for detection of enterovirus and rhinovirus RNAs in clinical specimen s and cell culture supernatants is described. RNA was extracted from s tool samples, throat swabs, nasopharyngeal aspirates, cerebrospinal fl uid, urine, and plasma with a commercial phenol-guanidinium-chloroform reagent and purified on a polysulfone membrane, on which the reverse transcriptase reaction was also done. Two sets of oligonucleotide prim ers from the 5' noncoding region of picornaviruses were selected for D NA amplification of 153-bp (enterovirus) and 120-bp (rhinovirus) regio ns. Double-stranded amplicons were digested into single strands with T 7 gene 6 exonuclease and quantitated by an assay using a europium-labe led probe, streptavidin- and biotinylated probe-coated microtitration wells, and time-resolved fluorometry. The sensitivity of the assay was about one template molecule when purified coxsackievirus A9 RNA was u sed. All enterovirus prototype strains, except echoviruses 22 and 23, and clinical isolates grown in cell culture or suckling mice were stro ngly positive by the enterovirus PCR-hybridization, as were selected p rototype strains and untyped isolates of rhinoviruses by the rhinoviru s PCR-hybridization. In a series of 100 clinical specimens tested, the results for 92 agreed with virus culture results. The detection metho d described will be useful in etiopathogenic studies on enteroviruses and rhinoviruses.